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Fig. 4 tfap2a and foxD3 are necessary for ASM specification. A) Fluorescent WISH analysis of foxC1a, lmx1b.1 and eya2 expression (magenta) in 32hpf embryos from tfap2asa2445/+ in-crosses. DNA was stained with DAPI (blue). Lateral images of volume projections of 3D confocal stacks from representative embryos are displayed. Proportion of embryos with corresponding expression phenotypes are displayed. In approximately 25% of embryos foxC1a, lmx1b.1 and eya2 expression was severely reduced. B) Fluorescent WISH analysis of foxC1a, lmx1b.1 and eya2 expression (magenta) in 32hpf embryos from foxD3sym1/+ in-crosses. DNA was stained with DAPI (blue). Lateral images of volume projections of 3D confocal stacks from representative embryos are displayed. Proportion of embryos with corresponding expression phenotypes are displayed. In approximately 25% of embryos foxC1a, lmx1b.1 and eya2 expression was severely reduced. C) Fluorescent WISH analysis of foxC1a, lmx1b.1 and eya2 expression (magenta) in 32hpf embryos from sox10cls/+ in-crosses. DNA was stained with DAPI (blue). Lateral images of volume projections of 3D confocal stacks from representative embryos are displayed. Proportion of embryos with corresponding expression phenotypes are displayed. All embryos examined displayed expected, wildtype, expression phenotypes. D) Fluorescent WISH analysis of foxC1a, lmx1b.1 and eya2 expression (magenta) in 32hpf embryos injected with golden crRNA or sox10 crRNA (CRISPR). DNA was stained with DAPI (blue). Lateral images of volume projections of 3D confocal stacks from representative embryos are displayed. Proportion of embryos with corresponding expression phenotypes are displayed. In both, golden and sox10 crRNA injected embryos expression of foxC1a, lmx1b.1 and eya2 was not affected. Scale bar = 100μm.